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In the American regions, Brazil accounts for 97% of visceral leishmaniasis (VL) cases, with a case fatality rate of approximately 10%. This study aimed to investigate the VL mortality distribution in Brazil and identify high-priority and high-risk areas for intervention strategies. This was an ecological study that analysed the spatial–temporal patterns of VL mortality in Brazilian municipalities. Age-standardised VL mortality rates from the Global Burden of Disease study from 2001 to 2018 were used. The distribution of mortality in the municipalities was assessed, and subsequently the Local Index of Spatial Autocorrelation (LISA) analysis was conducted to identify contiguous areas with high mortality rates. Scan analysis identified clusters of high spatial–temporal risks. The highest mortality rates and clusters were in municipalities located in the Northeast region and in the states of Tocantins and Roraima (North region), Mato Grosso do Sul (Central-West region), and Minas Gerais (Southeast region). According to LISA, there was an increase in the number of municipalities classified as high priority from the first 3-year period (n = 434) to the last 3-year period (n = 644). The spatio-temporal analysis identified 21 high-risk clusters for VL mortality. Areas with a high risk of VL mortality should prioritise preventing transmission, invest in early diagnosis and treatment, and promote the training of healthcare professionals. [ABSTRACT FROM AUTHOR]

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani–infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling–induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection. [ABSTRACT FROM AUTHOR]

In Europe, up to 70% of visceral leishmaniasis (VL) cases occurring in adults living with HIV. People living with HIV with VL co-infection often display persistent parasitemia, requiring chronic intermittent anti- Leishmania therapies. Consequently, frequent VL relapses and higher mortality rates are common in these individuals. As such, it is of paramount importance to understand the reasons for parasite persistence to improve infection management. To outline possible causes for treatment failure in the context of HIV-VL, we followed a person living with HIV-VL co-infection for nine years in a 12-month period. We characterized: HIV-related clinicopathological alterations (CD4+ T counts and viremia) and Leishmania -specific seroreactivity, parasitemia, quantification of pro-inflammatory cytokines upon stimulation and studied a Leishmania clinical isolate recovered during this period. The subject presented controlled viremia and low CD4+ counts. The subject remained PCR positive for Leishmania and also seropositive. The cellular response to parasite antigens was erratic. The isolate was identified as the first Leishmania infantum case with evidence of decreased miltefosine susceptibility in Portugal. Treatment failure is a multifactorial process driven by host and parasite determinants. Still, the real-time determination of drug susceptibility profiles in clinical isolates is an unexplored resource in the monitoring of VL. [ABSTRACT FROM AUTHOR]

Phlebotomine sand flies (Diptera: Phlebotominae) are vectors of human and animal pathogens, including Leishmania species protozoan parasites and viruses of the genus Phlebovirus. In Europe, visceral zoonotic leishmaniasis caused by Leishmania infantum , a deadly disease when left untreated, is endemic in southern countries, and dogs are the main reservoir hosts for human infection. Most phleboviruses cause asymptomatic infections or flu-like syndromes in humans, but Toscana phlebovirus can cause meningitis and encephalitis. These diseases are likely to re-emerge, posing a growing threat to public and animal health. Potential triggers include the movement of humans and dogs, increasing numbers of immunosuppressive conditions, climate change and other human-mediated environmental changes. An overview of the main epidemiological characteristics of the pathogens transmitted by sand flies in Europe and the potential triggers involved in their emergence and re-emergence are reviewed here. There is a need to implement mandatory notification of human and canine leishmaniases and human phleboviruses and coordinated epidemiological surveillance programmes at a European level, and to raise awareness among healthcare professionals and citizens about sand fly-borne diseases, following a One Health approach. [Display omitted] [ABSTRACT FROM AUTHOR]

For the past 15 years, trials of combination therapy options for visceral leishmaniasis have been conducted with the aim of identifying effective, and safe treatment regimens that were shorter than existing monotherapy regimens and could also prevent or delay the emergence of drug resistance. Although first-line treatment currently relies on combination therapy in east Africa, this is not true in Latin America owing to disappointing trial results, with lower than expected efficacy seen for the combination treatment group. By contrast, several effective combination therapy regimens have been identified through trials on the Indian subcontinent; yet, first-line therapy is still AmBisome monotherapy as the drug is part of a free donation programme and is highly effective in this region. Achieving a short all-oral combination treatment will require new chemical entities, several of which are currently under evaluation. Future studies should systematically include pharmacological substudies to ensure optimal dosing for all patient groups. To achieve maximal impact of new combination treatments, mechanisms to ensure drug availability and access after trials should be established. Enhancing the longevity of current and novel treatments will require effective systems for early detection of emerging drug resistance. [ABSTRACT FROM AUTHOR]

Leishmania infections are global, occurring in 98 countries and all World Health Organization (WHO) regions with 600 million to 1 billion people at risk of infection. Visceral leishmaniasis is associated with almost 20,000 reported deaths annually, with children under 5 years of age being at the greatest risk of mortality. Amongst WHO-recognised Neglected Tropical Diseases (NTDs), leishmaniasis is one of the most important in terms of mortality and morbidity. With an increasing global burden of disease and a growing threat from climate change, urbanisation and drug resistance, there remains an imperative to develop leishmaniasis vaccines. New tools to understand correlates of protection and to assess vaccine efficacy are being developed to ease the transition into larger scale efficacy trials or provide alternate routes to licensure. Early indications suggest a diverse portfolio of manufacturers exists in endemic countries with an appetite to develop leishmaniasis vaccines. This Vaccine Value Profile (VVP) provides a high-level, comprehensive assessment of the currently available data to inform the potential public health, economic, and societal value of leishmaniasis vaccines. The leishmaniasis VVP was developed by a working group of subject matter experts from academia, public health groups, policy organizations, and non-profit organizations. All contributors have extensive expertise on various elements of the leishmaniasis VVP and have collectively described the state of knowledge and identified the current gaps. The VVP was developed using only existing and publicly available information. [ABSTRACT FROM AUTHOR]

• Essential genes (Pyridoxal kinase and sterol alpha-14 demethylase) are drug targets. • Discerned the atomistic interactions of small molecules with target proteins through Molecular Docking and MD Simulations. • Non-bonded contacts and MM-PBSA calculations aided in deciphering nitazoxanide and artemisinin dynamic interactions with the target proteins. • This biocomputing study identified plausible drug inhibitors against essential leishmanial genes. Leishmania donovani , an obligatory intracellular flagellate pathogen, is the underlying cause of visceral leishmaniasis (VL), a fatal disease that poses a significant challenge to existing therapeutic approaches and leads to human mortality. In an endeavor to find an antileishmanial drug to combat VL, we aimed to assess the approved drug molecules against the specific drug targets of VL. In this study, a theoretical method was used to select two essential therapeutic targets (pyridoxal kinase [PK] and sterol alpha-14 demethylase [SDM]) which were present in both the data set of essential genes and drug target proteins. The selected PK and SDM proteins in L. donovani play pivotal roles as essential enzymes in the crucial vitamin B6 salvage and sterol biosynthesis pathways, respectively, leading to pathogenicity in humans. In addition to that drugs were gathered from the DrugBank and Drug Central databases and 325 (out of 4867) compounds having anti-parasitic properties were screened by PASS analysis. Consequently, three ligands (referred to as Lig_1, Lig_2, and Lig_3) were chosen based on their elevated Pa values, docking scores, and notable medicinal applications. Moreover, the result obtained from MD simulation suggests Lig_1 [Nitazoxanide (PubChem ID-41684)] does not affect the structural integrity of both targets. Additionally, evaluation of total binding energies by MMPBSA analysis showed stronger binding of Lig_1 with PK and SDM is -100.71 and -175.61 kJ/mol, respectively compared to others. As a whole, the methodology employed in this research involves the simultaneous identification of suitable protein targets and potential inhibitors. Through this investigation, we have demonstrated that compounds derived from a biocomputing approach exhibit interaction mechanisms as inhibitors against drug targets, offering a promising avenue for addressing VL. [ABSTRACT FROM AUTHOR]

Les leishmanioses sont des maladies tropicales négligées, causées par une quinzaine d'espèces d'un protozoaire flagellé du genre Leishmania et transmises par un moucheron hématophage, le phlébotome. Il existe trois principales formes cliniques : les leishmanioses cutanées, cutanéomuqueuses et viscérales selon la dissémination des parasites dans le système des phagocytes mononucléés. En 2021, les leishmanioses sont endémiques de 99 pays sur tous les continents habités hors Océanie et les dernières estimations font état de 600 000 à 1 million de cas de leishmanioses cutanées et de 50 000 à 90 000 cas de leishmanioses viscérales annuels. En France métropolitaine, les cas autochtones sont beaucoup moins fréquents que ceux d'importation. Le diagnostic de référence est la PCR sur sang pour les leishmanioses viscérales et la PCR sur prélèvement cutané pour les versions cutanées. Cet examen doit être associé à la sérologie dans le cadre des leishmanioses viscérales et cutanéomuqueuses. L'identification de l'espèce en cause est souhaitable dans les formes cutanées ou cutanéomuqueuses pour orienter la prise en charge thérapeutique. La complexité du diagnostic et du traitement peut rendre parfois nécessaire l'aide d'un centre spécialisé. Leishmaniasis is a neglected tropical disease caused by around fifteen species of a flagellate protozoan of the genus Leishmania, transmitted by a haematophagous sandfly. There are three main clinical forms: cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis, depending on the dissemination of the parasites in the mononuclear phagocyte system. In 2021, leishmaniasis is endemic in 99 countries on all inhabited continents excluding Oceania, and the latest estimates put the number of cases of cutaneous leishmaniasis at between 600,000 and 1 million, and the number of cases of visceral leishmaniasis at between 50,000 and 90,000 per year. In metropolitan France, native cases are much less frequent than imported cases. The reference diagnosis is PCR on blood for visceral leishmaniasis and PCR on skin samples for cutaneous leishmaniasis. This test must be combined with serology for visceral and mucocutaneous leishmaniasis. Identification of the species involved is recommended for cutaneous or mucocutaneous forms in order to guide therapeutic management. The complexity of diagnosing and treating leishmaniasis may sometimes require the assistance of a specialist centre. [ABSTRACT FROM AUTHOR]

COVID-19 has largely impacted the management of Visceral leishmaniasis (VL), like several other Neglected Tropical Diseases. The impact was particularly evident in Lower and Middle-Income countries where the already inadequate healthcare resources were diverted to managing the COVID-19 pandemic. Bangladesh achieved the elimination target for VL in 2016. To sustain this success, early diagnosis and treatment, effective vector control, and periodic surveillance are paramount. However, the specific control measures for VL in Bangladesh that were hampered during COVID-19 and their extent are unknown. This study aimed at identifying the gaps and challenges in the follow-up of treated VL patients by interviewing both the treated VL cases and their health service providers. We followed VL cases treated between 2019 and 2020 in five VL endemic subdistricts (upazilas) both retrospectively and prospectively to monitor clinical improvement, relapse, or other consequences. Moreover, interviews were conducted with the health service providers to assess the impact of COVID-19 on VL case detection, treatment, reporting, vector control operations, and logistic supply chain management. There was no added delay for VL diagnosis; however, VL treatment initiation and reporting time increased almost two-fold due to COVID-19. Indoor Residual Spraying activity was significantly hampered due to a shortage of insecticides. Out of 44 enrolled and treated VL patients, two relapsed (4.5 %), two developed Para Kala-Azar Dermal Leishmaniasis (4.5 %), and three (6.8 %) Post Kala-Azar Dermal Leishmaniasis (PKDL). The health service providers highlighted patients' unwillingness to visit the hospital, financial constraints, and distance from the hospitals as the main reasons for missed follow-up visits (20.5 %). Building good communication in the community, awareness schemes, and incentive-based approaches were suggested as possible solutions to mitigate these problems. Long-term follow-up is required for the early detection and management of VL relapse and PKDL cases. Effective vector control measures, capacity development, and identification of new VL hotspots are pivotal in the VL endemic regions to sustain the elimination goal. • The impact of the novel coronavirus (COVID-19) outbreak on visceral leishmaniasis (VL) case management and control services. • The importance of long-term follow-up of treated VL. • Factors responsible for missed post-treatment follow-up from the patients' and service providers' perspectives. • Recommendations by health service providers to increase post-treatment follow-up visits. • The findings of this study will help the national kala-azar elimination programme in future pandemic preparedness to sustain the VL elimination achievements. [ABSTRACT FROM AUTHOR]

Post kala-azar dermal leishmaniasis (PKDL) is the only dermal form of leishmaniasis that presents with hypopigmentation comparable with vitiligo, leprosy, and pitryasis versicolor. In the absence of an animal model, the mechanism(s) responsible for melanocyte loss/dysfunction in PKDL is/are poorly defined. Although the destruction of melanocytes is considered multifactorial, immune dysregulation is one of the key contributory players in this and other hypopigmentary dermatoses. Understanding of the complex interplay between immune cells and mediators, leading to the loss/destruction of melanocytes in PKDL, could translate into 'host-directed' chemotherapeutic strategies to prevent the stigma associated with hypopigmentation. Post kala-azar dermal leishmaniasis (PKDL), a heterogeneous dermal sequela of visceral leishmaniasis (VL), is challenging in terms of its etiopathogenesis. Hypopigmentation is a consistent clinical feature in PKDL, but mechanisms contributing to the loss of melanocytes remains poorly defined. Like other hypopigmentary dermatoses – for example, vitiligo, psoriasis, and leprosy – the destruction of melanocytes is likely a multifactorial phenomenon, key players being immune dysregulation and inflammation. This review focuses on immunological mechanisms responsible for the 'murder' of melanocytes, prime suspects at the lesional sites being CD8+ T cells and keratinocytes and their criminal tools being proinflammatory cytokines, for example, IFN-γ, IL-6, and TNF-α. Collectively, these may cause decreased secretion of melanocyte growth factors, loss/attenuation of cell adhesion molecules and inflammasome activation, culminating in melanocyte death. [ABSTRACT FROM AUTHOR]

The drug discovery pipeline for leishmaniasis and trypanosomiasis has been filling with novel chemical entities with known mechanisms of action. González et al. and Braillard et al. report a cytochrome bc 1 complex inhibitor as another promising preclinical candidate for visceral leishmaniasis (VL) and, in combination with benznidazole, for chronic Chagas' disease (CCD). [ABSTRACT FROM AUTHOR]

Post kala-azar dermal leishmaniasis (PKDL) is a skin disease that usually occurs among individuals with a past history of visceral leishmaniasis (VL). PKDL cases act as a reservoir of parasites and may play a significant role in disease transmission. Hence, prompt detection and complete treatment of PKDL cases are crucial for the control and elimination of VL. The purpose of this review was to highlight the barriers to effective control and prevention of VL/PKDL as well as potential solutions in India. Main obstacles are lack of knowledge about the disease and its vector, poor treatment-seeking behaviours, ineffective vector control measures, lack of confirmatory diagnostics in endemic areas, limited drug choices, treatment noncompliance among patients, drug resistance, and a lack of an adequate number of trained personnel in the health system. Therefore, in order to control and successfully eliminate VL in the Indian subcontinent, early detection of PKDL cases, improved diagnosis and treatment, raising awareness, and effective vector control mechanisms are necessary. [ABSTRACT FROM AUTHOR]

• Severe visceral leishmaniasis with non-ulcerated cutaneous leishmaniasis. • A Crithidia -related parasite was isolated from bone marrow aspirate and spleen. • Whole-genome sequencing of clinical isolates suggested a novel parasite species. • Clinical implications of this trypanosomatid co-infection remain unknown in visceral leishmaniasis. [Display omitted] We report a refractory and relapsed visceral leishmaniasis case in a male child patient followed from 2016 to 2020, whose clinical isolates from multiple relapses were analyzed at the genome level. To the best of our knowledge, it is the first report that both visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis have concomitantly manifested in the same patient. Importantly, sequence analysis revealed that the patient was co-infected with Leishmania infantum and a Crithidia -related parasite, which was previously found in a fatal case of visceral leishmaniasis from the same endemic region. [ABSTRACT FROM AUTHOR]

Organ transplantation can lead to human visceral leishmaniasis (VL) transmission in humans. This report aims to describe the possible complications related to an atypical course of VL after kidney transplantation. A 61-year-old man who suffered end-stage renal failure received a deceased donor kidney transplant after 2 years of hemodialysis. Tacrolimus, mycophenolate mofetil, and prednisolone were used for immunosuppressive therapy, and renal function remained stable for 2.5 years. He was referred to our hospital because of fever and malaise. Physical and radiological examinations showed mild splenomegaly and cervical and inguinal lymphadenopathy. Laboratory data showed bicytopenia, elevated C-reactive protein, serum creatinine, and non-nephrotic proteinuria. Bone marrow biopsy aspiration showed no abnormality. Polymerase chain reaction confirmed the diagnosis of Leishmania infantum. Anti-leishmanial therapy was initiated with liposomal amphotericin B for 2 weeks, and the patient became clinically stable. So far, there has been no evidence of clinical or biological relapse, and kidney function is stable. Considering that VL has become increasingly widespread in immunocompromised patients in endemic regions, especially in patients with transplants, it is crucial to screen and rule out VL as a cause of infection in these patients. The probability of this problem should be considered in every patient with a transplant in endemic and nonendemic areas. Furthermore, our study showed that through timely diagnosis using noninvasive methods and standard treatments, mortality caused by this disease can be properly prevented. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a tropical parasitic disease caused by Leishmania spp. They cause several presentations of illness ranging from cutaneous leishmaniasis to visceral leishmaniasis. The current arsenal of drugs to treat leishmaniasis is limited, and drug resistance further impedes the problem. Therefore, it is necessary to revisit the available information to identify an alternative or new target for treatment. The glycoprotein 63 (gp63), is a potential anti-leishmanial target that plays a significant role in host-pathogen interaction and virulence. Many studies are ongoing to develop gp63 inhibitors or use it as a vaccine target. In this review, we will discuss the potential of gp63 as a drug target. This review summarises the studies focusing on gp63 as a drug target and its inhibitors identified using in silico approaches. [Display omitted] • gp63 a potential drug and vaccine target. • Active site is conserved in all Leishmania spp. • May serve as pan drug target. • In silico studies identify its inhibitors. [ABSTRACT FROM AUTHOR]

A clear understanding of the accuracy of diagnostic tests is essential for the management of infections such as visceral leishmaniasis in endemic areas. Using both published and unpublished datasets from field diagnostic trials of visceral leishmaniasis in the past ten years, we show the potential effects of unrecognised sources of bias including work-up bias, referral bias, and reference test bias on the results. We outline why these biases, which can occur in diagnostic studies of any disease, can go unrecognised despite adherence to current STARD and QUADAS-2 guidelines. Using these examples and referring to others seen in studies of bacterial and viral infections, we make specific recommendations on how these biases might be avoided through specific steps in study design, study reporting, and the locations where studies are conducted. [ABSTRACT FROM AUTHOR]

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL. [Display omitted] • Endonuclease III (ENDO) was evaluated as vaccine candidate against L. infantum. • Saponin and polymeric micelles were used as Th1-type adjuvants. • Mice immunized with ENDO plus adjuvants mounted a specific Th1-type response. • Parasitological protection was reached using both vaccine compositions. • Endonuclease III induced in vitro immunogenicity in human cells. [ABSTRACT FROM AUTHOR]

Iron (Fe) sequestration is one of the most important strategies of the host to control the growth and survival of invading pathogens. Ferritin (Ft) plays a pivotal role in the sequestration mechanism of mammalian hosts by storing Fe. Recent evidence suggests that poly(rC)-binding proteins (PCBPs) act as chaperones for loading Fe into Ft. Incidentally, modulation of host PCBPs in respect to storing Fe in Ft during any infection remains unexplored. Among PCBPs, PCBP1 and PCBP2 are present in every cell type and involved in interacting with Ft for Fe loading. Leishmania donovani (LD) resides within macrophages during the mammalian stage of infection, causing life-threatening visceral leishmaniasis. Here, we reveal the ability of LD to cleave PCBP1 and PCBP2 in host monocytes/macrophages. LD cleaves PCBP1-FLAG into two fragments and PCBP2-FLAG into multiple fragments, thus affecting their in-teractions with Ft and resulting in decreased Fe loading into Ft. LD-derived culture supernatant or exosome-enriched fractions are also able to cleave PCBPs, suggesting involvement of a secreted protease of the parasite. Using an immune-depletion experiment and transgenic mutants, we confirmed the involvement of zinc-metalloprotease GP63 in cleaving PCBPs. We further revealed that by cleaving host PCBPs, Leishmania could inhibit Fe loading into Ft to accumulate available Fe for higher intracellular growth. This is the first report of a novel strategy adopted by a mammalian pathogen to interfere with Fe sequestration into Ft by cleaving chaperones for its survival advantage within the host. [ABSTRACT FROM AUTHOR]

[Display omitted] • Leishmania infantum is the only Leishmania species circulating in Italy. • In the Emilia-Romagna region, humans and dogs are infected by two distinct L. infantum strains. • For the first known time L. infantum infection was recorded in ruminants (roe deer). • Over 90% of L. infantum infections in deer are associated with the "human" strain. • This result suggests that these animals are a novel reservoir for human visceral leishmaniasis. Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum , responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region. [ABSTRACT FROM AUTHOR]

Dogs are the most common domestic reservoir of Leishmania infantum , making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL. [Display omitted] • Leishmania infantum -infected macrophages control parasites more efficiently when co-cultured with CD4+ and CD8+ T cells. • Heterologous co-culture from asymptomatic dogs has lower parasite burden than cultures from symptomatic dogs. • Co-culture system from dogs with CVL is a promising tool for screening immunotherapeutic agents for such disease. [ABSTRACT FROM AUTHOR]

• STAT1 ((Signal transducer and activator of transcription 1) serves as common factor upregulated in both VL and PKDL positive cases. • VLDLR (Very low density lipoprotein Receptor) exhibits significant suppression in PKDL and murine VL cases. • hsa-miR-30d-5p, hsa-miR-1226–3p, and hsa-miR-6891–3p regulates VLDLR for both VL and PKDL positive infection. Post Kala-azar dermal leishmaniasis (PKDL) arises as a significant dermal sequel following Visceral leishmaniasis (VL) caused by protozoan parasite Leishmania donovani (LD). PKDL acts as a significant constrain for VL elimination serving as a crucial reservoir for LD. PKDL patients exhibit depigmented macular and papular lesions on their skin, which results in social discrimination due to loss of natural skin color. Inflammatory reactions, prevalent in both VL and PKDL, potentially lead to tissue damage in areas harboring the parasite. Disruption of the immune-inflammasomal network not only facilitates LD persistence but also leads to the skin hypopigmentation seen in PKDL, impacting social well-being. Activation of inflammasomal markers like STAT1, NLRP1, NLRP3, AIM2, CASP11, and NLRP12 have been identified as a common host-defense mechanism across various Leishmania infections. Conversely, Leishmania modulates inflammasome activation to sustain its presence within the host. Nevertheless, in specific instances of Leishmania infection, inflammasome activation can worsen disease pathology by promoting parasite proliferation and persistence. This study encompasses recent transcriptomic analyses conducted between 2016 and 2023 on human and murine subjects afflicted with VL/PKDL, elucidating significant alterations in inflammasomal markers in both conditions. It offers a comprehensive understanding how these markers contribute in disease progression, drawing upon available literature for logical analysis. Furthermore, our analysis identifies validated miRNA network that could potentially disrupt this crucial immune-inflammasomal network, thereby offering a plausible explanation on how secreted LD-factors could enable membrane-bound LD, isolated from the host cytoplasm, to modulate cytoplasmic inflammasomal markers. Insights from this study could guide the development of host-directed therapeutics to impede transmission and address hypopigmentation, thereby mitigating the social stigma associated with PKDL. Schematic representation of key inflammasome related genes STAT1 and VLDLR, common between VL and PKDL positive cases from whole genome transcriptomics analysis of seven publicly available datasets and possible miRNAs networks regulating these inflammasomal markers. [Display omitted] [ABSTRACT FROM AUTHOR]

• The part of the insect used as a sample has an impact on the peptide/protein profile • Biological variables altered the profiles, generating identifiable diagnostic ions • When identifying sand flies with MALDI-TOF, biological factors must be considered • In the future, it will be possible to use MALDI-TOF to identify wild sand flies Sand flies are vectors of great public health importance, since they constitute a group of hematophagous insects responsible for etiological agents transmission of zoonotic diseases such a visceral leishmaniasis. In face of the expansion of these diseases, efficient control strategies are needed which depend on comprehending the sand fly eco-epidemiology. In this regard, MALDI-TOF mass spectrometry has been used for bacteria, fungi and yeast detection studies through peptide/protein profiles. However, little is known about interference of biological factors associated with vector ecology, such as blood meal preferences and even sand fly age on the peptide/protein profiles. Thus, the present study aimed to evaluate the differences in peptide/protein profiles of the sand fly Lutzomyia longipalpis , by means of MALDI-TOF, due to the sand fly's age, sex, blood meal source and Leishmania infantum infection. Sample preparation was made removing both head and last abdomen segments keeping the thorax, its appendices and the rest of the abdomen. Five specimens per pool were used to obtain peptide/protein extract of which 1 μL solution was deposited over 1 μL MALDI matrix dried. Characteristic spectra were analyzed using principal coordinate analysis as well as indicator species analysis to discriminate differences in sand flies's peptide/protein profile by sex, age, blood meal source and L. infantum infection. The results show that the evaluated variables produced distinct peptide/protein profiles, demonstrated by the identification of specific diagnostic ions. It was found that the interference of biological factors should be taken into account when using the MALDI-TOF analysis of sand fly species identification and eco-epidemiological applications in field studies. Based on our results, we believe that it is possible to identify infected specimens and the source of blood meal in a collection of wild sand flies, serving to measure infectivity and understand the dynamics of the vector's transmission chain. Our results may be useful for epidemiological studies that look at the ecology of sand flies and leishmaniasis, as well as for raising awareness of biological characteristics' impact on peptide/protein profiles in sand fly species identification. Experimental study design to evaluate the biological effects of age, blood feeding and Leishmania infantum infection in the sand fly species Lutzomyia longipalpis. [Display omitted] [ABSTRACT FROM AUTHOR]

• The study aims at exploring the potential bioactive compound from the extract of Northeastern plant Garcinia cowa belonging to Clusiaceae family having leishmanicidal activity against Leishmania donovani. • HR-LC-MS based metabolomic profiling study revealed presence of characteristic phytoconstituents. • Utilizing computational tools, modern day technique of molecular docking used for virtual screening of α-mangostin and β-mangostin for ideal lead molecule identification. • The xanthoid, α-mangostin was observed to have inhibitory activity on the viability of the promastigotes, axenic amastigotes as well as intracellular amastigotes. • The compound induces oxidative and nitrosative burst in the parasite resulting in onset of inflammatory response led by overproduction of nitric oxide. • The oxidative stress further induces cellular damage in form of nuclear fragmentation and mitochondrial dysfunction ultimately leading to apoptotic cell death of parasite, substantiating the anti-leishmanial effect of α-mangostin. Owing to the persistent number of parasitic deaths, Visceral leishmaniasis continues to haunt several economically weaker sections of India. The disease causes over 30,000 deaths and threatens millions annually on a global scale. The standard pentavalent antimonials, on the other hand, are associated with health adversities and disease relapse. The current study is focused on the search for the most potential natural bioactive phytocompound from the bark extract of the Northeastern Indian plant, Garcinia cowa , that shows potent anti-leishmanial properties. The High Resonance Liquid Chromatography followed by Mass Spectrometry (HR-LCMS) study followed by an in silico molecular docking using computational tools revealed that α-mangostin might potentially possess antiparasitic activity. To validate the anti-leishmanial efficacy of the compound, a cell viability assay was performed, which demonstrated the parasite-specific inhibitory activity of α-mangostin; with IC 50 values ranging from 4.95 - 7.37 µM against the different forms of Leishmania donovani parasite. The flow cytometric analysis of the phytocompound treated parasites indicated an oxidative and nitrosative stress-mediated apoptotic cell death in the parasites, by the suggestive surge in nuclear fragmentation and mitochondrial dysfunction. Simultaneously, a cytokine profiling study suggested approximate two-to-three-fold upregulated levels of pro-inflammatory cytokines post-compound treatment, which is predicted to actively contribute to parasite-killing. α-mangostin was also found to reduce the chances of parasite survival by inhibiting arginase enzyme activity, which in favorable conditions facilitates its sustenance. This study thereby substantiates that α-mangostin significantly possesses anti-leishmanial potentiality that can be developed into a cure for this infectious disease. [Display omitted] [ABSTRACT FROM AUTHOR]

• Our research investigated the genetic variations among CL, VL, and CanL strains in northern Morocco. • Molecular and genetic analyses uncovered a complex array of genetic variants in the L. infantum and L. tropica species. The number of these variants is 7 and 9 for L. tropica and L. infantum species, respectively. • The Discriminant Analysis of Principal Components (DAPC) revealed significant genetic divergences among L. infantum strains. This suggests the influence of species origin on their genetic makeup. This study investigated nine provinces in northern Morocco and collected 275 skin scraping, 22 bone marrow aspirates, and 89 fine needle aspirations from suspected cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) patients and potentially infected dogs. Molecular analysis using ITS1 RFLP PCR and RT-PCR revealed a higher prevalence of L. infantum (66.18 %; χ2 = 28.804; df = 1; P-value = 8.01e−08) than L. tropica in skin scraping, with L. infantum being the sole causative agent for both VL and canine leishmaniasis. L. infantum was predominantly found in most provinces, while L. tropica was relatively more dominant in Taza Province. Discriminant Analysis of Principal Components (DAPC) revealed distinct clustering between L. tropica and the other three species. However, no small subset of SNPs could clearly differentiate between Infantum_CL, Infantum_VL, and CanL, as they likely share a significant genetic background. The high rate of L. infantum could be attributed to the abundance of sand fly species transmitting VL. In Taza Province, Phlebotomus sergenti, responsible for anthroponotic CL, is the most abundant species. DNA sequencing demonstrated sequence heterogeneity in L. infantum (variants 1-9) and L. tropica (variants 1-7). Phylogenetic analysis showed a distinct separation between L. tropica and L. infantum strains, with an overlap among L. infantum strains isolated from cutaneous, visceral, and canine cases, and dogs serving as the central population for L. infantum. [ABSTRACT FROM AUTHOR]

Leishmaniases, caused by Leishmania parasites, are widespread and pose significant health risks globally. Visceral leishmaniasis (VL) is particularly prevalent in Brazil, with high morbidity and mortality rates. Traditional treatments, such as pentavalent antimonials, have limitations due to toxicity and resistance. Therefore, exploring new compounds like lectins is crucial. Concanavalin A (ConA) has shown promise in inhibiting Leishmania growth. This study aimed to evaluate its leishmanicidal effect on L. infantum promastigotes and understand its mechanism of action. In vitro tests demonstrated inhibition of promastigote growth when treated with ConA, with IC 50 values ranging from 3 to 5 μM over 24–72 h. This study suggests that ConA interacts with L. infantum glycans. Additionally, ConA caused damage to the membrane integrity of parasites and induced ROS production, contributing to parasite death. Scanning electron microscopy confirmed morphological alterations in treated promastigotes. ConA combined with the amphotericin B (AmB) showed synergistic effects, reducing the required dose of AmB, and potentially mitigating its toxicity. ConA demonstrated no cytotoxic effects on macrophages, instead stimulating their proliferation. These findings reinforce that lectin exhibits promising leishmanicidal activity against L. infantum promastigotes, making ConA a potential candidate for leishmaniasis treatment. • ConA has anti-promastigote activity. • ConA interacts with leishmania glycans. • ConA induces membrane and cell wall damage and ROS overproduction. • ConA increases the action of amphotericin B. [ABSTRACT FROM AUTHOR]

Leishmaniasis, caused by the protozoan parasite poses a significant health burden globally. With a very few specific drugs, increased drug resistance it is important to look for drug repurposing along with the identification of pre-clinical candidates against visceral leishmaniasis. This study aims to identify potential drug candidates against visceral leishmaniasis by targeting leishmanial MAP kinases and screening FDA approved protein kinase inhibitors. MAP kinases were identified from the Leishmania genome. 12 FDA approved protein kinase inhibitors were screened against Leishmania MAP kinases. Binding affinity, ADME and toxicity of identified drug candidates were profiled. The anti-proliferative effects and mechanism of action were assessed in Leishmania , including changes in cell morphology, flagellar length, cell cycle progression, reactive oxygen species (ROS) generation, and intra-macrophage parasitic burden. 23 MAP kinases were identified from the Leishmania genome. Sorafenib and imatinib emerged as repurposable drug candidates and demonstrated excellent anti-proliferative effects in Leishmania. Treatment with these inhibitors resulted in significant changes in cell morphology, flagellar length, and cell cycle arrest. Furthermore, sorafenib and imatinib promoted ROS generation and reduced intra-macrophage parasitic burden, and elicited anti-leishmanial activity in in vivo experimental VL models. Collectively, these results imply involvement of MAP kinases in infectivity and survival of the parasite and can pave the avenue for repurposing sorafenib and imatinib as anti-leishmanial agents. These findings contribute to the exploration of new treatment options for visceral leishmaniasis, particularly in the context of emerging drug resistance. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Innovative peptides SPR imunossensor for canine leishmaniasis diagnosis. • Comparison of two immunogenic synthetic peptides as bioreceptors. • This simple label-free methodology allowed fast, sensitive, and selective analysis. • Via PCA was able to observe that even smaller times of detection can be explored. • Excellent ability to discriminate between positive and negative canine real samples. A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor's analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis (CVL), an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol L-1), and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs' samples. For this reason, the PEP13 immunosensor was used to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases. [ABSTRACT FROM AUTHOR]

The obligate intracellular parasite Leishmania binds several receptors to trigger uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. A series of signaling pathways in host cells, which are critical for establishment and persistence of infection, are activated during Leishmania internalization. Thus, preventing Leishmania uptake by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery mediating promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibiting host MEK1/2 or ERK1/2 leads to inefficient amastigote uptake. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used to treat cancer, reduces disease severity and parasite burden in Leishmania -infected mice, even if it is started after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis. [ABSTRACT FROM AUTHOR]

• Superparamagnetic iron oxide nanoparticles (MNPs) were synthesized for provide cheaper and high performance Leishmaniasis serodiagnosis. • Synthetic peptide it was validated in a nanomagnetic ELISA for Leishmaniasis serodiagnosis. • Nanomagnetic ELISA results has proven an advantageous technique, a less time-consuming process with optimized detection. Serodiagnosis methods have been used as platforms for diagnostic tests for many diseases. Due to magnetic nanoparticles' properties to quickly detach from an external magnetic field and particle size effects, these nanomaterials' functionalization allows the specific isolation of target analytes, enhancing accuracy parameters and reducing serodiagnosis time. Superparamagnetic iron oxide nanoparticles (MNPs) were synthesized and functionalized with polyethylene glycol (PEG) and then associated with the synthetic Leishmaniosis epitope. This nano-peptide antigen showed promising results. Regarding Tegumentary leishmaniasis diagnostic accuracy, the AUC was 0.8398 with sensibility 75% (95CI% 50.50 - 89.82) and specificity 87.50% (95CI% 71.93 - 95.03), and Visceral leishmaniasis accuracy study also present high performance, the AUC was 0.9258 with sensibility 87.50% (95CI% 63.98 - 97.78) and specificity 87.50% (95CI% 71.93 - 95.03). Our results demonstrate that the association of the antigen with MNPs accelerates and improves the diagnosis process. MNPs could be an important tool for enhancing serodiagnosis. [Display omitted] [ABSTRACT FROM AUTHOR]

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras in Mesocricetus auratus hamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease. [ABSTRACT FROM AUTHOR]

• Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis, which can be fatal. • Visceral leishmaniasis treatment may be toxic, and resistance has been reported. • There are currently no vaccines available for humans. • Transgenic L. infantum could be an excellent vaccine candidate. • Prime-boosted mice have shown maximized protection. The etiologic agents of visceral leishmaniasis are Leishmania infantum and Leishmania donovani. Despite the variety of drugs available to treat leishmaniasis, most lead to serious adverse effects, and resistance to these drugs has been reported. Currently, no leishmaniasis vaccine is available for humans. That is why the group developed transgenic L. infantum promastigote lines, which express toxic proteins after differentiation into amastigotes. That is why group developed the pFL-AMA plasmid and transfected it into L. Infantum promastigotes. This plasmid was expressed only in the amastigote form of the parasite. Sequences encoding toxic proteins (active bovine trypsin and egg avidin) were inserted in this plasmid, and the transfected parasites died after the differentiation process. In this study, two immunization protocols were performed in BALB/c mice: prime and prime-boost immunization prior to challenge with the wild-type L. infantum (WT). The parasite burdens in the spleen, liver, and bone marrow were evaluated to verify immunological protection. Histopathological analysis of the spleen and liver and the humoral immune response were also performed. The data showed that the parasite burden was reduced in prime-boosted mice in the spleen, liver, and bone marrow, indicating that mice immunized with two doses of the transfected parasites were satisfactorily protected. High levels of IgG, IgG1, and IgG2a antibodies were observed, as well as the presence of anti-inflammatory cytokine Interleukine-10 and pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN – γ) suggesting a Th1/Th2 mix response, in addition to the presence of multinucleated giant cells in the spleen and lymphocyte infiltration in the liver. Therefore, L. infantum transfected with a toxic plasmid is an excellent vaccine candidate against visceral leishmaniasis and the application of a boost before the challenge promotes greater protection against WT L. infantum infection. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients. • Leishmania donovani infection impairs uptake of cholesterol. • Retinoic acid (RA) increases the expressions of genes involved in cholesterol uptake. • RA restores the levels of cellular cholesterol in Leishmania donovani infected mφ. • Treatment with RA reduces the parasite burden in infected mφ. [ABSTRACT FROM AUTHOR]

• Visceral leishmaniasis is a serious complication, especially in immunosuppressed patients, and is uncommon after liver transplantation. • The case report highlights the challenge of early diagnosis of visceral leishmaniasis in liver transplant recipients with atypical manifestations. • The aim of the report is to emphasize the suspicion of visceral leishmaniasis in symptomatic patients from emerging countries and to question the benefit of including protozoan screening in liver transplant donors and recipients in endemic areas. Leishmaniasis is a disease caused by a protozoan and transmitted by sandfly species in several emerging countries. Visceral leishmaniasis is a serious complication, especially in immunosuppressed patients, and is uncommon after liver transplantation. We report the case of a 48-year-old female patient who underwent liver transplantation owing to polycystic liver disease. Six months after the procedure, she was hospitalized with diarrhea, acute kidney failure, and leukopenia. She had been off steroids for 3 months and was taking mycophenolate and tacrolimus. She had already been treated for cytomegalovirus, which was negative on admission. During hospitalization, fever, splenomegaly, ascites, and pancytopenia appeared. Serology for Leishmania by indirect immunofluorescence was negative. Then, bone biopsy and molecular testing for Leishmania diagnosed it as visceral leishmaniasis. Amphotericin therapy was initiated with resolution of fever after 4 days of treatment and gradual recovery from pancytopenia. This case highlights the challenge of early diagnosis of visceral leishmaniasis in liver transplant recipients with diarrhea and leukopenia, which can be caused by immunosuppression or more prevalent viral diseases. Late onset of fever, splenomegaly, and a first negative serologic test also made early diagnosis difficult. The aim of the report is to emphasize the suspicion of visceral leishmaniasis in symptomatic patients from emerging countries and to question the benefit of including protozoan screening in liver transplant donors and recipients in endemic areas. [ABSTRACT FROM AUTHOR]

Herein, carboxymethyl chitosan (CMC) grafted lipid nanoformulations were facilely prepared by thin-film hydration method as a highly efficient biocompatible anti-leishmanial carrier encapsulating amphotericin B (AmB). Nanoformulations were characterized for their physicochemical characteristics wherein TEM analysis confirmed the spherical structure, whereas FTIR analysis revealed the conjugation of CMC onto nanoformulations and confirmed the free state of AmB. Furthermore, the wettability study confirmed the presence of CMC on the surface of nanoformulations attributed to the enhanced hydrophilicity. Surface hydrophilicity additionally contributes towards consistent mucin retention ability for up to 6 h, superior mucoadhesiveness, and hence enhanced bioavailability. The proposed nanoformulations with high encapsulation and drug loading properties displayed controlled drug release in the physiological microenvironment. In vitro, antileishmanial results showed an astounding 97% inhibition in amastigote growth. Additionally, in vivo studies showed that treatment with nanoformulations significantly reduced the liver parasitic burden (93.5%) without causing any toxicity when given orally. [Display omitted] [ABSTRACT FROM AUTHOR]

• Flame synthesis and characterisation of CoFe 2 O 4 -C60NP. • Synthesis and characterisation of A2 peptide antigen using LCMS and HPLC. • A2 peptide antigen decorated CoFe 2 O 4 -C60NP immunoelectrode. • Immunosensor with 30.34 fg/mL LOD at a linear range of 10−10 to 10−1 µg/mL. • Label-free electrochemical detection of A2-antibodies in spiked blood serum. Diagnosis of Visceral Leishmaniasis is challenging due to the shared clinical features with malaria, typhoid, and tuberculosis. A CoFe 2 O 4 -C60 nanocomposite-based immunosensor decorated with a sensitive A2 peptide antigen was fabricated to detect anti-A2 antibodies for application in visceral leishmaniasis diagnosis. The flame-synthesised nanocomposite was characterised using Fourier Transform Infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), Scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX), Raman spectroscopy and electrochemical impedance spectroscopy (EIS) techniques. N terminated specific A2 peptide epitope antigen (NH 2 -QSVGPLSVGP-OH) was synthesised and characterised by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Using EDC/NHS, A2 peptide antigen (Apg) was immobilised on the CoFe 2 O 4 -C60-modified electrode. The performance of the immunosensor, Apg-CoFe 2 O 4 -C60NP/GCE, was evaluated by testing its ability to detect varying concentrations of anti-A2 antibody solution in PBS and spiked serum with 1 mM [Fe(CN) 6 ]3−/4− in 0.01 M PBS (pH 7.4) as supporting electrolyte. using differential pulse voltammetry. The immunosensor showed excellent reproducibility and a linear range of 10−10–10−1 µg/mL, with an experimental detection limit of 30.34 fg/mL. These results suggest that the fabricated sensor has great potential as a tool for diagnosing visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

In visceral and mucocutaneous leishmaniasis, humoral immune response can reflect disease severity and parasite burden. Cutaneous leishmaniasis (CL) in Sri Lanka is caused by a usually visceralizing parasite, Leishmania donovani. We assessed the parasite burden (relative quantity—RQ) in 190 CL patients using quantitative real-time PCR (qPCR—with primers designed for this study) and smear microscopy, then correlated it with clinical parameters and IgG response. RQ of parasite DNA was determined with human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The qPCR sensitivity was tested with serially diluted DNA from cultured L. donovani parasites. Smears were assigned a score based on number of parasites per high power field. Data from previous studies were used for comparison and correlation; nested Internal Transcribed Spacer 1 (ITS1) PCR as reference standard (RS) and IgG antibody titers to the Leishmania rKRp42 antigen as the immune response. The qPCR amplified and quantified 86.8% of the samples while demonstrating a fair and significant agreement with ITS1-PCR and microscopy. Parasite burden by qPCR and microscopy were highly correlated (r = 0.76; p = 0.01) but showed no correlation with the IgG response (r = 0.056; p = 0.48). Corresponding mean RQs of IgG titers grouped by percentiles, showed no significant difference (p = 0.93). Mean RQ was higher in early lesions (p = 0.04), decreased with lesion size (p = 0.12) and slightly higher among papules, nodules and wet ulcers (p = 0.72). Our study established qPCR's efficacy in quantifying parasite burden in Sri Lankan CL lesions but no significant correlation was observed between the parasite burden and host IgG response to the Leishmania rKRP42 antigen. [Display omitted] • qPCR effectively quantified parasite burden in Sri Lankan cutaneous leishmaniasis. • Microscopic- and qPCR-based quantification of parasite burden correlated well. • Parasite burden and anti-rKRP42 IgG level showed no correlation in cutaneous form. • Parasite burden was higher in early lesions, decreasing with lesion size. [ABSTRACT FROM AUTHOR]

Leishmania , a protozoan parasite, is responsible for the occurrence of leishmaniasis, a disease that is prevalent in tropical regions. Visceral Leishmaniasis (VL), also known as kala-azar in Asian countries, is one of the most significant forms of VL, along with Cutaneous Leishmaniasis (CL) and Mucocutaneous Leishmaniasis (ML). Management of this condition typically entails the use of chemotherapy as the sole therapeutic option. The current treatments for leishmaniasis present several drawbacks, including a multitude of side effects, prolonged treatment duration, disparate efficacy across different regions, and the emergence of resistance. To address this urgent need, it is imperative to identify alternative treatments that are both safer and more effective. The identification of appropriate pharmacological targets in conjunction with biological pathways constitutes the initial stage of drug discovery. In this review, we have addressed the key metabolic pathways that represent potential pharmacological targets as well as prominent treatment options for leishmaniasis. • Target-based drug discovery. • Essential metabolic pathways in Leishmania like sterol biosynthesis, trypanothione route, glycolytic, Protein kinases, arginase and polyamine biosynthesis, trypanothione synthase and trypanothione reductase, topoisomerases, purine salvage, mannose biosynthesis etc. that are possible pharmacological targets. • Some important drugs for the treatment of leishmaniasis. [ABSTRACT FROM AUTHOR]

The present work aimed to develop and evaluate AmB-loaded nano-emulsion (AmB-NE) which will augment the solubility of AmB and lead to enhanced anti-leishmanial activity. The composition of AmB-NE was optimized by systematic screening followed by DoE-extreme vertices mixture design. The optimized NE revealed mean droplet size and PDI of 44.19 ± 5.5 nm, 0.265 ± 0.0723, respectively. The NE could efficiently encapsulate AmB with drug content and efficiency 83.509 ± 0.369% and 81.659 ± 0.013%, respectively. The presence of cholesterol and stearyl amine retarded the release (P < 0.0001) of AmB significantly compared to AmB suspension. The AmB-NE and pure AmB suspension demonstrated the IC 50 of 0.06309 μg/mL and 0.3309 μg/mL against L. donovani promastigotes after 48 h incubation. The formulation was robust at all exaggerated stability conditions such as freeze-thaw and centrifugation. These findings indicate that AmB-NE is an attractive approach to treat visceral leishmaniasis with improved activity. [Display omitted] • Amphotericin-loaded nanoemulsion was developed via extreme vertices mixture design. • Average size and polydispersity index was 44.19 ± 5.5 nm, 0.265 ± 0.072, respectively. • Inclusion of cholesterol and stearylamine imparted sustained release character. • Nanoemulsion caused significantly higher activity against L. donovani promastigotes. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis. [Display omitted] • HTS, design, optimization gave substituted 2-aminoquinazolines as L. donovani inhibitors. • Scaffold-hopping gave leads with improved potency against L. donovani Vs hit 1. • Lead compounds exhibited better potency compared to Miltefosine. • Lead compounds displayed a good selectivity index against THP-1 macrophages. • Microwave-assisted ArSN reaction at C2 position of quinazoline derivatives. [ABSTRACT FROM AUTHOR]

• Growth of Leishmania parasites is inhibited by clotrimazole. • Clotrimazole effect was reversed by ergosterol supplementation. • Clotrimazole modulates cell membrane potential and its permeability. • Generates mitochondrial superoxide and causes mitochondrial depolarization. • Clotrimazole treated cells are arrested at sub G 0 stage of cell cycle. Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC 50) of 35.75 ± 1.06 μM, 12.75 ± 0.35 μM and 73 ± 1.41 μM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G 0 phase suggesting apoptotic mode of cell death. [Display omitted] [ABSTRACT FROM AUTHOR]

• There is a positive association between VL and El Niño, when compared to La Niña. • VL incidence was significant in Brazil's central and Northeast and Colombia's Northeast regions. • There is an impressive% of area suitable for the vector Lu. longipalpis in the Amazon region. • The potential distribution of Lu. longipalpis expanded especially in Roraima, Pará, Acre, and Mato Grosso. Visceral Leishmaniasis (VL) is the most severe of the three forms of Leishmaniasis. In the Americas, Brazil and Colombia present more than 90 % of the cases in the region. Our aim in this research was to estimate the association of the incidence rate of Visceral Leishmaniasis with the following environmental variables: the percentage of area suitable for the vector Lutzomyia longipalpis, t he episodes of La Niña and El Niño, the Brazilian and Colombian biomes. Epidemiological data were obtained from the Brazilian Notifiable Diseases Information System and the Colombian National Public Health Surveillance System. Environmental data were downloaded from the NASA Giovanni web app, the Modis Sensor database, and the meteorological agencies of Australia, Japan, and the United States of America. Records of the presence of Lu. longipalpis were obtained from public databases and previous studies. As a result, the incidence per 10,000 inhabitants with LEBS for each El Niño-Southern Oscillation (ENSO) episode showed the largest values during El Niño 2015–2016, mainly in Brazil's Northeast and Central regions and the Northeast region of Colombia. Compared with the Neutral 2012–2014 episode, the episodes of El Niño 2015–2016 and La Niña 2010–2011 showed an average increase in the monthly incidence rate of VL, and the average increase was higher during El Niño 2015–2016 (aIRR = 2.304 vs.1.453) We found a positive association between the incidence rate of VL and the El Niño 2015–2016 episode and an impressive% of area suitable for the vector Lu. longipalpis in the Amazon region.An increase of 1 % in the area suitable for the vector Lu. longipalpis leads to an average rise of 0.8 % in the monthly incidence rate of VL. Our study shows a possible association between VL incidence and ENSO, with the most considerable incidence rates observed during El Niño 2015–2016 in Brazil's Northeast and Central regions and the Northeast region of Colombia. The present study is very important to better understand the Visceral Leishmaniasis transmission dynamics. [ABSTRACT FROM AUTHOR]

• The age of mice directly affects leishmania parasitemia. • Use of the LaAg vaccine intranasally helps in the process of controlling parasite in young and elderly mice. • The process of controlling L. infantum infection is directly related to the expression of inhibitory receptors such as PD-1 and KLRG-1. There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+ T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis. [Display omitted] [ABSTRACT FROM AUTHOR]

• Visceral leishmaniasis can occur due to co-infection with several Leishmania spp. • Co-infection in visceral leishmaniasis changes our understanding of the disease. • Several trypanosomatids can be identified per sample using this protocol. • Amplicon-based next-generation sequencing is a cost-effective method in molecular epidemiological studies. Visceral leishmaniasis (VL) is a mammalian protozoal disease propagated in the Americas by female phlebotomine sandflies, mainly caused by Leishmania infantum. However, in recent years, cases of VL caused by different Leishmania species, such as L. amazonensis and L. colombiensis, have been reported in the continent. This study used an amplicon-based next-generation sequencing approach to identify VL aetiologic species using high-depth sequencing targeting a region on the Heat Shock Protein 70 gene. In this first approach, six samples from five patients diagnosed with VL were selected and analysed to identify DNA of Leishmania spp. All samples harboured DNA of L. infantum ; five samples were found to be co-infected with other Leishmania spp. or with Trypanosoma cruzi , and just one sample was mono-infected with L. infantum. This study demonstrates the usefulness of this methodology to identify trypanosomatid co-infections in clinical samples, which presents an interesting study panorama considering their biological, clinical and epidemiological implications. [Display omitted] [ABSTRACT FROM AUTHOR]

• Treatment with Nano Sbv or Anti IL-10R increase the IFN-γ producing T-CD4 lymphocytes in the initial post-treatment times. • The CVL treatment with Nano Sbv or Anti IL-10R restore the lymphoproliferative capacity in the initial post-treatment times. • Treatment does not improve the immunological, clinical and parasitological parameters at the end of the trial follow-up. This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum , displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum. [ABSTRACT FROM AUTHOR]

[Display omitted] • Immunogenicity and prophylactic potential of rLdTCP1γ was evaluated against VL. • rLdTCP1γ caused strong LTT response & NO production in cured animal compared to SLD. • rLdTCP1γ +BCG exhibited potent prophylactic efficacy against VL infection. • rLdTCP1γ +BCG elicited both antigen specific cellular and humoral immunity. • LdTCP1γ+BCG immunization resulted in 2.5 folds increased survival of hamsters. Visceral leishmaniasis (VL) is a chronic tropical disease responsible for devastating epidemics worldwide. Though current treatment relies on drugs, the emergence of resistance, toxic side-effects, and strenuous administration has led to an ineffective remedy. Hence, vaccination remains an alternative and desirable approach for VL control. Though extensive research on anti-leishmanial vaccine candidates has been carried out in past decades, presence of an effective molecule is still missing. In the present study, we have evaluated the immunogenicity and prophylactic potential of a recombinant T-complex protein-1 gamma subunit of L. donovani (rLdTCP1γ), against VL in hamster model. The antigen exhibited in vitro stimulation of lymphoproliferative and NO response in miltefosine and amphotericin B treated hamsters depicting its immunotherapeutic/immunogenic nature. Immunization with rLdTCP1γ revealed a strong protective response against experimental VL as indicated by reduced parasite load in the spleen of immunized group compared to infected control. The immunized animals gained body weight and exhibited significant reduction in the spleen and liver weight as compared to infected controls on days 60, 90, 120 post-challenge. A substantial augmentation of cell-mediated immune response as depicted by an increased lymphocyte proliferation, nitric oxide production, DTH responses and increased levels of IgG2 was observed in rLdTCP1γ immunized hamsters. The Th1 stimulatory potential, imparted by the antigen, was found to be intensified in the presence of adjuvant Bacillus Calmette-Guérin (BCG). The efficacy was further assisted by an upregulated mRNA transcript of Th1 induced cytokines (IL-12, IFN-γ and TNFα) and downregulation of IL-4 and IL-10. The results are thus suggestive of rLdTCP1γ having the potential of a strong vaccine candidate against VL. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a parasitic disease caused by different Leishmania species. L. infantum is found in the Mediterranean area. It usually causes visceral or cutaneous leishmaniasis, but rarely mucosal leishmaniasis (ML). A 62-year-old man with metastatic non-small-cell lung carcinoma visited the outpatient clinic because of a painful and swollen tongue. Initially, oral candidiasis was suspected and patient was unsuccessfully treated accordingly. Subsequently, a biopsy from the tongue was taken. Histology of the tongue biopsy showed an inflammation with histiocytes and Leishmania amastigotes. Molecular analysis determined these parasites as L. donovani complex. Based on the patient's travel history, ML caused by L. infantum was diagnosed. ML is an unusual presentation of L. infantum. ML is not only caused by Leishmania species endemic in Latin America, but also should be considered in the differential diagnosis for European patients. A biopsy of the affected location is needed to confirm the diagnosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phlebotomus argentipes is a sole vector of Leishmania donovani in the Indian subcontinent. • 40% of humans in the study area have IgG antibodies against P. argentipes saliva. • A correlation was found between IgG responses against P. argentipes saliva and rPagSP06. • rPagSP06 is a valid antigen to measure human exposure to P. argentipes. Phlebotomus argentipes is a predominant vector of Leishmania donovani , the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes , sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti- P. argentipes saliva IgG responses. The anti-rPagSP06 and anti- P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti- P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area. [ABSTRACT FROM AUTHOR]